In the following discussion certain articles and methods are described for background and introductory purposes. Nothing contained herein is to be construed as an “admission” of prior art. Applicant expressly reserves the right to demonstrate, where appropriate, that the articles and methods referenced herein do not constitute prior art under the applicable statutory provisions.
Antibodies have emerged as important biological pharmaceuticals because they (i) exhibit exquisite binding properties that can target antigens of diverse molecular forms, (ii) are physiological molecules with desirable pharmacokinetics that make them well tolerated in treated humans and animals, and (iii) are associated with powerful immunological properties that naturally ward off infectious agents. Furthermore, established technologies exist for the rapid isolation of antibodies from laboratory animals, which can readily mount a specific antibody response against virtually any foreign substance not present natively in the body.
In their most elemental form, antibodies are composed of two identical heavy (H) chains that are each paired with an identical light (L) chain. The N-termini of both H and L chains consist of a variable domain (VH and VL, respectively) that together provide the paired H-L chains with a unique antigen-binding specificity. The exons that encode the antibody VH and VL domains do not exist in the germ-line DNA. Instead, each VH exon is generated by the recombination of randomly selected V, D, and J gene segments present in the H chain locus (Igh; see schematic of the mouse Igh locus in FIG. 1); likewise, individual VL exons are produced by the chromosomal rearrangements of randomly selected V and J gene segments in a light chain locus. The canine genome contains two alleles that can express the H chain (one allele from each parent), two alleles that can express the kappa (κ) L chain, and two alleles that can express the lambda (λ) L chain. There are multiple V, D, and J gene segments at the H chain locus as well as multiple V and J gene segments at both L chain loci. Downstream of the J genes at each immunoglobulin (Ig) locus exists one or more exons that encode the constant region of the antibody. In the heavy chain locus, exons for the expression of different antibody classes (isotypes) also exist. In canine animals, the encoded isotypes are IgM, IgD, IgG1, IgG2, IgG3, IgG4, IgE, and IgA. Polymorphic variants (referred to as allotypes) also exist among canine strains for IgG2, IgE and IgA and are useful as allelic markers.
During B cell development, gene rearrangements occur first on one of the two homologous chromosomes that contain the H chain variable gene segments. The resultant VH exon is then spliced at the RNA level to the exons that encode the constant region of the H chain (CH). Subsequently, the VJ rearrangements occur on one L chain allele at a time until a functional L chain is produced, after which the L chain polypeptides can associate with the H chain homodimers to form a fully functional B cell receptor for antigen (BCR).
The genes encoding various canine (e.g., the domestic dog and wolf) and mouse immunoglobulins have been extensively characterized, although the sequence and annotation of the canine Ig loci in the genome databases is not yet complete. Priat, et al., describe whole-genome radiation mapping of the dog genome in Genomics, 54:361-78 (1998), and Bao, et al., describe the molecular characterization of the VH repertoire in Canis familiaris in Veterinary Immunology and Immunopathology, 137:64-75 (2010). Blankenstein and Krawinkel describe the mouse variable heavy chain region in Eur. J. Immunol., 17:1351-1357 (1987). The generation of transgenic animals—such as mice having varied immunoglobulin loci—has allowed the use of such transgenic animals in various research and development applications, e.g., in drug discovery and basic research into various biological systems. For example, the generation of transgenic mice bearing human immunoglobulin genes is described in International Application WO 90/10077 and WO 90/04036. WO 90/04036 describes a transgenic mouse with an integrated human immunoglobulin “mini” locus. WO 90/10077 describes a vector containing the immunoglobulin dominant control region for use in generating transgenic animals.
Numerous methods have been developed for modifying the mouse endogenous immunoglobulin variable region gene locus with, e.g., human immunoglobulin sequences to create partly or fully-human antibodies for drug discovery purposes. Examples of such mice include those described in, e.g., U.S. Pat. Nos. 7,145,056; 7,064,244; 7,041,871; 6,673,986; 6,596,541; 6,570,061; 6,162,963; 6,130,364; 6,091,001; 6,023,010; 5,593,598; 5,877,397; 5,874,299; 5,814,318; 5,789,650; 5,661,016; 5,612,205; and 5,591,669. However, many of the fully humanized immunoglobulin transgenic mice exhibit suboptimal antibody production because B cell development in these mice is severely hampered by inefficient V(D)J recombination, and by inability of the fully human antibodies/BCRs to function optimally with mouse signaling proteins. Other humanized immunoglobulin transgenic mice, in which the mouse coding sequence have been “swapped” with human sequences, are very time consuming and expensive to create due to the approach of replacing individual mouse exons with the syntenic human counterpart.
The use of antibodies that function as drugs is not necessarily limited to the prevention or therapy of human disease. Companion animals such as dogs suffer from some of the same afflictions as humans, e.g., cancer, atopic dermatitis and chronic pain. Monoclonal antibodies targeting CD20, IgE and Nerve Growth Factor, respectively, are already in veterinary use as for treatment of these conditions. However, before clinical use these monoclonal antibodies, which were made in mice, had to be caninized, i.e., their amino acid sequence had to be changed from mouse to dog, in order to prevent an immune response in the recipient dogs. Based on the foregoing, it is clear that a need exists for efficient and cost-effective methods to produce canine antibodies for the treatment of diseases in dogs. More particularly, there is a need in the art for small, rapidly breeding, non-canine mammals capable of producing antigen-specific canine immunoglobulins. Such non-canine mammals are useful for generating hybridomas capable of large-scale production of canine monoclonal antibodies.
In accordance with the foregoing object, transgenic nonhuman animals are provided which are capable of producing an antibody with canine V regions.